LOCUS       62056_13390        8194 bp DNA     circular SYN 07-JUL-2019
DEFINITION  Cloning vector pKill1, complete sequence.
ACCESSION   MK756312
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8194)
  AUTHORS   Neil K, Allard N, Jordan D, Rodrigue S.
  TITLE     Assembly of large mobilizable genetic cargo by double recombinase 
            operated insertion of DNA (DROID)
  JOURNAL   Plasmid (2019) In press
  PUBMED    31247227
REFERENCE   2  (bases 1 to 8194)
  AUTHORS   Neil K, Rodrigue S.
  TITLE     Direct Submission
  JOURNAL   Submitted (04-APR-2019) Biology, University of Sherbrooke, 2500 
            Boulevard de l'Universite, Sherbrooke, Quebec J1K 2R1, Canada
REFERENCE   3  (bases 1 to 8194)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Plasmid 
            (2019) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (04-APR-2019) Biology, University of Sherbrooke, 2500 Boulevard de 
            l'Universite, Sherbrooke, Quebec J1K 2R1, Canada"
COMMENT     ##Assembly-Data-START##
            Assembly Method       :: ApE v. 2018-05-04
            Sequencing Technology :: Putative Sequence
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..8194
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..58
                     /label=attPbxb1
                     /note="attPbxb1"
     rep_origin      539..761
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             809..1756
                     /label=Rep101(Ts)
                     /note="temperature-sensitive version of the RepA protein
                     needed for replication with the pSC101 origin (Armstrong et
                     al., 1984)"
     protein_bind    2192..2239
                     /label=FRT
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     regulatory      2267..2348
                     /label=apFAB391
                     /note="apFAB391"
                     /regulatory_class="terminator"
     regulatory      2349..2398
                     /label=PN25
                     /note="PN25"
                     /regulatory_class="promoter"
     misc_feature    2399..2418
                     /label=spacer
                     /note="spacer"
     misc_RNA        2419..2494
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      2499..2529
                     /label=B1006U10
                     /note="B1006U10"
                     /regulatory_class="terminator"
     regulatory      2727..2776
                     /regulatory_class="promoter"
     CDS             2865..3656
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
     CDS             4088..8191
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
ORIGIN
        1 gtcgtggttt gtctggtcaa ccaccgcggt ctcagtggtg tacggtacaa accccgaccg
       61 acagtaagac gggtaagcct gttgatgata ccgctgcctt actgggtgca ttagccagtc
      121 tgaatgacct gtcacgggat aatccgaagt ggtcagactg gaaaatcaga gggcaggaac
      181 tgctgaacag caaaaagtca gatagcacca catagcagac ccgccataaa acgccctgag
      241 aagcccgtga cgggcttttc ttgtattatg ggtagtttcc ttgcatgaat ccataaaagg
      301 cgcctgtagt gccatttacc cccattcact gccagagccg tgagcgcagc gaactgaatg
      361 tcacgaaaaa gacagcgact caggtgcctg atggtcggag acaaaaggaa tattcagcga
      421 tttgcccgag cttgcgaggg tgctacttaa gcctttaggg ttttaaggtc tgttttgtag
      481 aggagcaaac agcgtttgcg acatcctttt gtaatactgc ggaactgact aaagtagtga
      541 gttatacaca gggctgggat ctattctttt tatctttttt tattctttct ttattctata
      601 aattataacc acttgaatat aaacaaaaaa aacacacaaa ggtctagcgg aatttacaga
      661 gggtctagca gaatttacaa gttttccagc aaaggtctag cagaatttac agatacccac
      721 aactcaaagg aaaaggacta gtaattatca ttgactagcc catctcaatt ggtatagtga
      781 ttaaaatcac ctagaccaat tgagatgtat gtctgaatta gttgttttca aagcaaatga
      841 actagcgatt agtcgctatg acttaacgga gcatgaaacc aagctaattt tatgctgtgt
      901 ggcactactc aaccccacga ttgaaaaccc tacaaggaaa gaacggacgg tatcgttcac
      961 ttataaccaa tacgttcaga tgatgaacat cagtagggaa aatgcttatg gtgtattagc
     1021 taaagcaacc agagagctga tgacgagaac tgtggaaatc aggaatcctt tggttaaagg
     1081 ctttgagatt ttccagtgga caaactatgc caagttctca agcgaaaaat tagaattagt
     1141 ttttagtgaa gagatattgc cttatctttt ccagttaaaa aaattcataa aatataatct
     1201 ggaacatgtt aagtcttttg aaaacaaata ctctatgagg atttatgagt ggttattaaa
     1261 agaactaaca caaaagaaaa ctcacaaggc aaatatagag attagccttg atgaatttaa
     1321 gttcatgtta atgcttgaaa ataactacca tgagtttaaa aggcttaacc aatgggtttt
     1381 gaaaccaata agtaaagatt taaacactta cagcaatatg aaattggtgg ttgataagcg
     1441 aggccgcccg actgatacgt tgattttcca agttgaacta gatagacaaa tggatctcgt
     1501 aaccgaactt gagaacaacc agataaaaat gaatggtgac aaaataccaa caaccattac
     1561 atcagattcc tacctacata acggactaag aaaaacacta cacgatgctt taactgcaaa
     1621 aattcagctc accagttttg aggcaaaatt tttgagtgac atgcaaagta agtatgatct
     1681 caatggttcg ttctcatggc tcacgcaaaa acaacgaacc acactagaga acatactggc
     1741 taaatacgga aggatctgag gttcttatgg ctcttgtatc tatcagtgaa gcatcaagac
     1801 taacaaacaa aagtagaaca actgttcacc gttacatatc aaagggaaaa ctgtccatat
     1861 gcacagatga aaacggtgta aaaaagatag atacatcaga gcttttacga gtttttggtg
     1921 catttaaagc tgttcaccat gaacagatcg acaatgtaac agatgaacag catgtaacac
     1981 ctaatagaac aggtgaaacc agtaaaacaa agcaactaga acatgaaatt gaacacctga
     2041 gacaacttgt tacagctcaa cagtcacaca tagacagcct gaaacaggcg atgctgctta
     2101 tcgaatcaaa gctgccgaca acacgggagc cagtgacgcc tcccgtgggg aaaaaatcat
     2161 ggcaattctg gaagaaatag cgctttcagc cgaagttcct attccgaagt tcctattctc
     2221 tagaaagtat aggaacttcg tgcactgatt aagcattggt aacaggtcgg tcagtttcac
     2281 ctgatttacg taaaaacccg cttcggcggg tttttgcttt tggaggggca gaaagatgaa
     2341 tgactgtctc ataaaaaatt tatttgcttt caggaaaatt tttctgtata atagattctc
     2401 gcaagatgtg gcgtgttagt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
     2461 ttatcaactt gaaaaagtgg caccgagtcg gtgcttttaa aaaaaaaacc ccgcgcgggg
     2521 ttttttttta caagtctcag tgtgctgaac ccgggctgta gttcgtcaaa tgatggccac
     2581 ttttccgacg gtgctgaccc cggatgaatg tcagctactg ggctatctgg acaagggaaa
     2641 acgcaagcgc aaagagaaag caggtagctt gcagtgggct tacatggcga tagctagact
     2701 gggcggtttt atggacagca agcgaaccgg aattgccagc tggggcgccc tctggtaagg
     2761 ttgggaagcc ctgcaaagta aactggatgg ctttcttgcc gccaaggatc tgatggcgca
     2821 ggggatcaag atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     2881 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     2941 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     3001 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc
     3061 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     3121 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     3181 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     3241 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     3301 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     3361 cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg
     3421 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     3481 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     3541 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     3601 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     3661 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     3721 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     3781 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccagcttcaa
     3841 aagcgctttt atcagccata aaacaatact taatactata gaatgataac aaaataaact
     3901 actttttaaa agaattttgt gttataatct atttattatt aagtattggg taatattttt
     3961 tgaagagata ttttgaaaaa gaaaaattaa agcatattaa actaatttcg gaggtcatta
     4021 aaactattat tgaaatcatc aaactcatta tggatttaat ttaaactttt tattttagga
     4081 ggcaaaaatg gataagaaat actcaatagg cttagatatc ggcacaaata gcgtcggatg
     4141 ggcggtgatc actgatgaat ataaggttcc gtctaaaaag ttcaaggttc tgggaaatac
     4201 agaccgccac agtatcaaaa aaaatcttat aggggctctt ttatttgaca gtggagagac
     4261 agcggaagcg actcgtctca aacggacagc tcgtagaagg tatacacgtc ggaagaatcg
     4321 tatttgttat ctacaggaga ttttttcaaa tgagatggcg aaagtagatg atagtttctt
     4381 tcatcgactt gaagagtctt ttttggtgga agaagacaag aagcatgaac gtcatcctat
     4441 ttttggaaat atagtagatg aagttgctta tcatgagaaa tatccaacta tctatcatct
     4501 gcgaaaaaaa ttggtagatt ctactgataa agcggatttg cgcttaatct atttggcctt
     4561 agcgcatatg attaagtttc gtggtcattt tttgattgag ggagatttaa atcctgataa
     4621 tagtgatgtg gacaaactat ttatccagtt ggtacaaacc tacaatcaat tatttgaaga
     4681 aaaccctatt aacgcaagtg gagtagatgc taaagcgatt ctttctgcac gattgagtaa
     4741 atcaagacga ttagaaaatc tcattgctca gctccccggt gagaagaaaa atggcttatt
     4801 tgggaatctc attgctttgt cattgggttt gacccctaat tttaaatcaa attttgattt
     4861 ggcagaagat gctaaattac agctttcaaa agatacttac gatgatgatt tagataattt
     4921 attggcgcaa attggagatc aatatgctga tttgtttttg gcagctaaga atttatcaga
     4981 tgctatttta ctttcagata tcctaagagt aaatactgaa ataactaagg ctcccctatc
     5041 agcttcaatg attaaacgct acgatgaaca tcatcaagac ttgactcttt taaaagcttt
     5101 agttcgacaa caacttccag aaaagtataa agaaatcttt tttgatcaat caaaaaacgg
     5161 atatgcaggt tatattgatg ggggagctag ccaagaagaa ttttataaat ttatcaaacc
     5221 aattttagaa aaaatggatg gtactgagga attattggtg aaactaaatc gtgaagattt
     5281 gctgcgcaag caacggacct ttgacaacgg ctctattccc catcaaattc acttgggtga
     5341 gctgcatgct attttgagaa gacaagaaga cttttatcca tttttaaaag acaatcgtga
     5401 gaagattgaa aaaatcttga cttttcgaat tccttattat gttggtccat tggcgcgtgg
     5461 caatagtcgt tttgcatgga tgactcggaa gtctgaagaa acaattaccc catggaattt
     5521 tgaagaagtt gtcgataaag gtgcttcagc tcaatcattt attgaacgca tgacaaactt
     5581 tgataaaaat cttccaaatg aaaaagtact accaaaacat agtttgcttt atgagtattt
     5641 tacggtttat aacgaattga caaaggtcaa atatgttact gaaggaatgc gaaaaccagc
     5701 atttctttca ggtgaacaga agaaagccat tgttgattta ctcttcaaaa caaatcgaaa
     5761 agtaaccgtt aagcaattaa aagaagatta tttcaaaaaa atagaatgtt ttgatagtgt
     5821 tgaaatttca ggagttgaag atagatttaa tgcttcatta ggtacctacc atgatttgct
     5881 aaaaattatt aaagataaag attttttgga taatgaagaa aatgaagata tcttagagga
     5941 tattgtttta acattgacct tatttgaaga tagggagatg attgaggaaa gacttaaaac
     6001 atatgctcac ctctttgatg ataaggtgat gaaacagctt aaacgtcgcc gttatactgg
     6061 ttggggacgt ttgtctcgaa aattgattaa tggtattagg gataagcaat ctggcaaaac
     6121 aatattagat tttttgaaat cagatggttt tgccaatcgc aattttatgc agctgatcca
     6181 tgatgatagt ttgacattta aagaagacat tcaaaaagca caagtgtctg gacaaggcga
     6241 tagtttacat gaacatattg caaatttagc tggtagccct gctattaaaa aaggtatttt
     6301 acagactgta aaagttgttg atgaattggt caaagtaatg gggcggcata agccagaaaa
     6361 tatcgttatt gaaatggcac gtgaaaatca gacaactcaa aagggccaga aaaattcgcg
     6421 agagcgtatg aaacgaatcg aagaaggtat caaagaatta ggaagtcaga ttcttaaaga
     6481 gcatcctgtt gaaaatactc aattgcaaaa tgaaaagctc tatctctatt atctccaaaa
     6541 tggaagagac atgtatgtgg accaagaatt agatattaat cgtttaagtg attatgatgt
     6601 cgatcacatt gttccacaaa gtttccttaa agacgattca atagacaata aggtcttaac
     6661 gcgttctgat aaaaatcgtg gtaaatcgga taacgttcca agtgaagaag tagtcaaaaa
     6721 gatgaaaaac tattggagac aacttctaaa cgccaagtta atcactcaac gtaagtttga
     6781 taatttaacg aaagctgaac gtggaggttt gagtgaactt gataaagctg gttttatcaa
     6841 acgccaattg gttgaaactc gccaaatcac taagcatgtg gcacaaattt tggatagtcg
     6901 catgaatact aaatacgatg aaaatgataa acttattcga gaggttaaag tgattacctt
     6961 aaaatctaaa ttagtttctg acttccgaaa agatttccaa ttctataaag tacgtgagat
     7021 taacaattac catcatgccc atgatgcgta tctaaatgcc gtcgttggaa ctgctttgat
     7081 taagaaatat ccaaaacttg aatcggagtt tgtctatggt gattataaag tttatgatgt
     7141 tcgtaaaatg attgctaagt ctgagcaaga aataggcaaa gcaaccgcaa aatatttctt
     7201 ttactctaat atcatgaact tcttcaaaac agaaattaca cttgcaaatg gagagattcg
     7261 caaacgccct ctaatcgaaa ctaatgggga aactggagaa attgtctggg ataaagggcg
     7321 agattttgcc acagtgcgca aagtattgtc catgccccaa gtcaatattg tcaagaaaac
     7381 agaagtacag acaggcggat tctccaagga gtcaatttta ccaaaaagaa attcggacaa
     7441 gcttattgct cgtaaaaaag actgggatcc aaaaaaatat ggtggttttg atagtccaac
     7501 ggtagcttat tcagtcctag tggttgctaa ggtggaaaaa gggaaatcga agaagttaaa
     7561 atccgttaaa gagttactag ggatcacaat tatggaaaga agttcctttg aaaaaaatcc
     7621 gattgacttt ttagaagcta aaggatataa ggaagttaaa aaagacttaa tcattaaact
     7681 acctaaatat agtctttttg agttagaaaa cggtcgtaaa cggatgctgg ctagtgccgg
     7741 agaattacaa aaaggaaatg agctggctct gccaagcaaa tatgtgaatt ttttatattt
     7801 agctagtcat tatgaaaagt tgaagggtag tccagaagat aacgaacaaa aacaattgtt
     7861 tgtggagcag cataagcatt atttagatga gattattgag caaatcagtg aattttctaa
     7921 gcgtgttatt ttagcagatg ccaatttaga taaagttctt agtgcatata acaaacatag
     7981 agacaaacca atacgtgaac aagcagaaaa tattattcat ttatttacgt tgacgaatct
     8041 tggagctccc gctgctttta aatattttga tacaacaatt gatcgtaaac gatatacgtc
     8101 tacaaaagaa gttttagatg ccactcttat ccatcaatcc atcactggtc tttatgaaac
     8161 acgcattgat ttgagtcagc taggaggtga ctga
//
